A urinary organic acids profile can be utilized as an effective screening tool for analyzing abnormality of nutrient metabolism. By using these metabolic markers in conjunction with one another, it helps in understanding how individual nutrient metabolism is executed and to determine where there may be imbalances in the metabolic cycle. In this study, we developed a rapid quantification method of 20 urinary organic acids by HPLC-mass spectrometry. A pre-analytical process of organic acid extraction from a urine sample is crucial in this methodology. The process was accomplished by liquid–liquid extraction followed by strong anion exchange. Compared with previous methods, this method greatly reduces the analysis time and allows for the simultaneous quantification of 20 organic acids within 10 min for the first time. This methodology enabled us to analyze urine samples collected from 34 Chinese children. The abnormalities of some urinary organic acids were found in this group, which revealed evidence of functional inadequacy of specific nutrients. The preliminary data in this study confirmed the suitability of the method for rapid and accurate quantification of the target organic acids in urine samples.